Bottom Line:
The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within.These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter.Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments.

fig04: Principal coordinate plots of unweighted UniFrac distances between all sequenced samples (A), Pseudomyrmex flavicornis samples (B), Pseudomyrmex nigrocinctus samples (C), and Cephalotes varians samples (D). Colors correspond to different species in panel A and different colonies in B–D. Adults and larvae are represented as open and filled symbols, respectively.

Mentions:
No pattern was apparent between extraction protocols in PCoA plots using unweighted UniFrac distances between samples (Fig. 4), but differences between species, colonies, and life stages were clear. Neither ANOSIM nor PERMANOVA analyses found significant differences between extraction methodologies when all samples were included (P > 0.1; Fig. 4), regardless of the beta diversity metric used. The lack of significant effect held true when only samples within individual species were compared (P > 0.1; Fig. 4). However, Species hosted significantly different communities according to all beta diversity metrics (ANOSIM, PERMANOVA, P = 0.001; Fig. 4). Within species, colonies hosted significantly different communities (P < 0.005; Fig. 4). While extractions from three abdomens and one abdomen were not significantly different for P. nigrocinctus or Cephalotes varians (P > 0.1), there were significant differences between the larval and adult extractions within P. flavicornis (P = 0.001; Fig. 4). The results from all statistical tests of beta diversity differences can be found in Table S4.

fig04: Principal coordinate plots of unweighted UniFrac distances between all sequenced samples (A), Pseudomyrmex flavicornis samples (B), Pseudomyrmex nigrocinctus samples (C), and Cephalotes varians samples (D). Colors correspond to different species in panel A and different colonies in B–D. Adults and larvae are represented as open and filled symbols, respectively.

Mentions:
No pattern was apparent between extraction protocols in PCoA plots using unweighted UniFrac distances between samples (Fig. 4), but differences between species, colonies, and life stages were clear. Neither ANOSIM nor PERMANOVA analyses found significant differences between extraction methodologies when all samples were included (P > 0.1; Fig. 4), regardless of the beta diversity metric used. The lack of significant effect held true when only samples within individual species were compared (P > 0.1; Fig. 4). However, Species hosted significantly different communities according to all beta diversity metrics (ANOSIM, PERMANOVA, P = 0.001; Fig. 4). Within species, colonies hosted significantly different communities (P < 0.005; Fig. 4). While extractions from three abdomens and one abdomen were not significantly different for P. nigrocinctus or Cephalotes varians (P > 0.1), there were significant differences between the larval and adult extractions within P. flavicornis (P = 0.001; Fig. 4). The results from all statistical tests of beta diversity differences can be found in Table S4.

Bottom Line:
The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within.These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter.Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments.